Identification Of Fibroblast Growth Factor5as A Target Of Let-7B:Implication In The Growth Of Alpaca Fibre

MicroRNAs are very small endogenous RNA molecules that are essential regulators of skin morphogenesis.Let-7b has been implicated in animal hair growth by binding its target genes.Fibroblast Growth Factor5(FGF5) may be involved in the hair growth cycle.In this study,combining previous reports with bioinformatics analysis to indetify potential let-7b target gene,we construct the dual-luciferase recombinant vector which contains3’-UTR of FGF5gene and let-7b over-expression vector.293T cells were treated with recomnination vector and let-7b over-expression vector,then to evaluate the activity of luciferase.To investigate the interaction between microRNA and target gene involved in Alpaca fibre growth,we transfected Alpaca fibroblasts with let-7b over-expression vector to detect the expression level of FGF5gene and its receptor fgfrl.Methods:Dual-luciferase Reporter Assay System was used to evaluate the luciferase activity of cotransfected with recombinant vetor and let-7b over-expression vector. Real-time PCR, western blot and immunocytochemical were used to investigate the expression level of gene FGF5and its receptor fgfrl.Results:(1)3’-UTR of FGF5gene was successfully cloned into pmirGLO vector.The luciferase activity ratio of293T cells cotransfected with recombinant vetor and let-7b over-expression vector was2.548times than control groups.Prelimimary evidence showed that let-7b could regulate the expression of FGF5.(2)We transfected let-7b over-expression vetor into Alpaca fibroblasts then to explore the expression level of its target gene FGF5and receptor for FGF5by Real-time PCR and western blot.The results showed that the FGF5mRNA quantity of transfected groups was0.287times than control groups,and the fgfrl mRNA quantity was2.251times(P<0.01).The protein level of FGF5in transfected groups was remarkably lower than control groups(P<0.05).The average relative gray value of FGF5protein and fgfrlprotein were0.224±0.019and0.952±0.152..The immunocytochemical results showed that expression domain and area of FGF5were chaged in transfected cells groups.In conclusion,let-7b may be involved in the growth of Alpaca fibre through inducing down-regulation of FGF5.